TYPE Original Research
PUBLISHED 18 September 2023
OPEN ACCESS
EDITED BY
Boris Klempa,
Slovak Academy of Sciences, Slovakia
REVIEWED BY
Kanisht Batra,
Lala Lajpat Rai University of Veterinary
and Animal Sciences, India
Niraj Kumar Singh,
Bihar Animal Sciences University, India
*CORRESPONDENCE
Xin Cao,
xinc@jlau.edu.cn
Chunfeng Wang,
wangchunfeng@jlau.edu.cn
†
These authors have contributed equally
to this work
RECEIVED 10 May 2023
ACCEPTED 25 August 2023
PUBLISHED 18 September 2023
CITATION
Kanyema MM, Cheng M, Luo J, Lu M,
Xing X, Sun Y, Wang J, Lu Y, Shi C,
Zeng Y, Yang G, Cao X and Wang C
(2023), Comprehensive codon usage
analysis of the African Swine Fever Virus.
Acta Virol. 67:11562.
doi: 10.3389/av.2023.11562
COPYRIGHT
© 2023 Kanyema, Cheng, Luo, Lu, Xing,
Sun, Wang, Lu, Shi, Zeng, Yang, Cao and
Wang. This is an open-access article
distributed under the terms of the
Creative Commons Attribution License
(CC BY). The use, distribution or
reproduction in other forums is
permitted, provided the original
author(s) and the copyright owner(s) are
credited and that the original
publication in this journal is cited, in
accordance with accepted academic
practice. No use, distribution or
reproduction is permitted which does
not comply with these terms.
Acta Virologica
DOI 10.3389/av.2023.11562
Comprehensive codon usage
analysis of the African Swine
Fever Virus
Makoye Mhozya Kanyema 1,2,3,4,5†, Mingyang Cheng 1,2,3,4†,
Jiawei Luo 1,2,3,4, Mei Lu 1,2,3,4, Xinyuan Xing 1,2,3,4, Yu Sun 1,2,3,4,
Junhong Wang 1,2,3,4, Yiyuan Lu 1,2,3,4, Chunwei Shi 1,2,3,4,
Yan Zeng 1,2,3,4, Guilian Yang 1,2,3,4, Xin Cao 1,2,3,4* and
Chunfeng Wang 1,2,3,4*
1
College of Veterinary Medicine, Jilin Agricultural University, Changchun, China, 2Jilin Provincial Key
Laboratory of Animal Microecology and Healthy Breeding, Jilin Agricultural University, Changchun,
China, 3Jilin Provincial Engineering Research Center of Animal Probiotics, Jilin Agricultural University,
Changchun, China, 4Key Laboratory of Animal Production and Product Quality Safety of Ministry of
Education, Jilin Agricultural University, Changchun, China, 5Department of Agricultural Sciences,
Mwalimu Julius K.Nyerere University of Agriculture and Technology, Mara, Tanzania
The non-uniform usage of synonymous codons occurs in genomes of all
organisms, including DNA and RNA viruses. The preferential selection of a
codon at the expense of other synonymous codons within the same group is
known as Codon Usage Bias. The understanding of this bias assists in unveiling
the factors driving molecular evolution, as defined by the selection-mutation-
drift theory. According to this model, molecular evolution is predominantly
driven by mutation, natural selection, and genetic drift. Nevertheless, elements
like nucleotide composition, gene length, and protein secondary structure also
contribute to this process. Comprehensive genomic analyses that highlight the
codon usage preference of the African Swine Fever Virus (ASFV) are infrequent.
ASFV, a hemorrhagic and highly contagious viral disease, almost invariably
results in 100% fatality among infected pigs and wild boars. This study,
therefore, embarked on a thorough examination of codon usage patterns in
ASFV’s complete genomic sequences, an endeavor of great relevance to
molecular evolution studies, complex transmission models, and vaccine
research. For an exhaustive evaluation of ASFV’s whole-genome codon
usage, we used parameters like ENC, RSCU, and CAI. A Principal
Component Analysis was carried out to reaffirm the interconnected RSCU
lineages based on the continent, and their evolutionary relationships were
later elucidated through phylogenetic tree construction. ASFV emerged as a
low-biased codon user (ENC = 52.8) that is moderately adapted to its host. Its
genome has a high AT composition (64.05%), suggesting the impact of
Abbreviations: ASFV, African Swine Fever Virus; CAI, Codon Adaptation Index; CSC, Chinese
Scholarship Council; CUB, Codon Usage Bias; DNA, Deoxyribose Nucleic Acid; ENC, Effective
Number of Codons; GRAVY, Grand Average of Hydropathy; IQR, Interquartile Range; MAFFT,
Multiple Alignment using Fast Fourier Transform; MAS, Multiple Sequence Alignment; MEGA,
Molecular Evolutionary Genetics Analysis; ML, Maximum Likelihood; NWK, Newick; ORF, Opening
Reading Frame; PCA, Principal Component Analysis; PR2, Chargaff’s second parity rule; R, Range;
RCDI, Relative Codon Deoptimization Index; RNA, Ribose Nucleic Acid; RSCU, Relative Synonymous
Codon Usage; SiD, Similarity Index.
Published by Frontiers
Institute of Virology
01 Biomedical Research Center, Slovak Academy of Sciences
Kanyema et al. 10.3389/av.2023.11562

mutational pressure on genomic evolution. However, neutrality plot analysis

revealed natural selection’s slight supremacy over mutational pressure. The low
codon bias (>45) implies ASFV’s diverse usage of synonymous codons within a
given codon family, allowing for effective translation and subsequent successful
viral replication cycles. Its moderate adaptation (CAI = 0.56) permits the virus to
infect a range of hosts, including reservoirs such as warthogs and bush pigs. To
the best of our knowledge, this is the pioneering report providing a
comprehensive examination of ASFV’s complete genomic sequences.
Consequently, research focusing on viral gene expression and regulation,
gene function prediction, parasite-host interaction, immune dysfunction,
and drug and vaccine design may find this report to be a valuable resource.

KEYWORDS

ASFV, codon usage bias, whole genome, continental lineages, phylogenic tree

Introduction diverse frequencies. Synonymous codons are substitute codons in

African Swine Fever (ASF) constitutes a virulent, infectious
hemorrhagic fever afflicting both domestic pigs and wild boars.
This deadly disease originates from the African Swine Fever
Virus (ASFV), the solitary DNA arbovirus and exclusive member
of the Asfivirus genus, nested within the Asfarviridae family
(Dixon et al., 2012; Jia et al., 2017; Teklue et al., 2020). Depending
on the ASFV strain, morbidity and mortality rates may escalate to
100%, making ASF a severe obstacle for global pig farming and,
consequently, worldwide food and nutrition security (Zhang
et al., 2022). ASFV is a double-stranded DNA virus ranging
from 170 to 193 kbp in length and encompassing between
151 and 167 open reading frames depending on the strain.
The virus presents a complex, multi-layered icosahedral virion
structure (Jia et al., 2017; Wang et al., 2021; Zheng et al., 2022). It
shares characteristics with other large nucleocytoplasmic DNA
viruses such as poxvirus and iridovirus (Iyer et al., 2001; Loh
et al., 2009).
Through nucleotide sequencing of the B646L gene, which
encodes the p72 major capsid protein, twenty-four (I to XXIV)
ASFV genotypes have been identified, all found in Africa. ASF
remains enzootic in sub-Saharan Africa and Sardinia, Italy (Sang
et al., 2020). However, it has migrated beyond these traditional
confines, infiltrating regions such as Caucasia, the European
Union, Asia, Oceania, and South America, and has recently
resurfaced in North America’s Haiti and the Dominican
Republic (Alkhamis et al., 2018; Ata et al., 2022; Ayanwale
et al., 2022; Chen et al., 2022). In 2018, ASF penetrated
China, the world’s leading pork producer, decimating
extensive pig populations and jeopardizing global food and
protein security. Since there are no treatments or vaccines for
ASF, the primary means of containment are quarantine, culling,
and rigorous movement restrictions (Salguero, 2020; Urbano and
Ferreira, 2020).
Codon Usage Bias (CUB) refers to the phenomenon wherein
synonymous codons within a codon family are employed at
the same group that offer a different coding sequence for a certain
amino acid (Zhang et al., 2011; Deb et al., 2021a). The genetic
code’s degeneracy facilitates this, meaning that a single amino
acid can be coded by multiple codons within the same family
(Nasrullah et al., 2015; Tian et al., 2018). This redundancy in the
genetic code is crucial in modulating the efficacy and precision of
protein synthesis while preserving the amino acid sequence of the
protein. Apart from methionine and tryptophan, this degeneracy
enables the remaining 18 amino acids to be encoded by 61 triplet
codons (Nasrullah et al., 2015; Yao et al., 2020).
Numerous DNA and RNA viruses exhibit CUB, with some,
such as the Hepatitis A Virus, showing significant bias (ENC =
39.78) (Andrea et al., 2011; Bera et al., 2017). In contrast, other
viruses, such as PDCoV (ENC = 52.69) (Peng et al., 2022), SARS-
CoV-2 (ENC = 45.38) (Hou, 2020), PEDV (ENC = 48.04) (Yu
et al., 2021a), CHIKV (ENC = 55.56) (Butt et al., 2014), ZIKV
(ENC = 53.32) (Wang et al., 2016), Classical Swine Fever Virus
(ENC = 51.7) (Tao et al., 2009), H7N9 Influenza A Virus (ENC =
51) (Sun et al., 2020), and Bluetongue (ENC = 57.9) (Yao et al.,
2020), display weak codon usage bias.
Several factors can potentially influence codon usage
patterns, including mutational pressure, natural selection,
genetic drift, gene length, nucleotide and amino acid
composition, secondary protein structure, replication, and
transcription factors, the hydrophobicity and hydrophilicity of
the protein, and environmental conditions (Butt et al., 2014;
Zhang et al., 2018). However, under the selection-mutation-
genetic drift model, natural selection, mutational pressure, and
genetic drift primarily account for variations in codon usage
among diverse organisms (Deb et al., 2021a). For many RNA and
DNA viruses, including Foot-and-Mouth Virus, Herpes Virus,
and Rubella Virus, mutational bias is more pronounced than
natural selection. Conversely, natural selection is instrumental in
the evolution of certain viruses, such as those in the Parvoviridae
family, Zika, Henipa, and Influenza A viruses. Therefore, despite
mutational pressure’s ongoing significance as an evolutionary

Published by Frontiers
Institute of Virology
Acta Virologica 02 Biomedical Research Center, Slovak Academy of Sciences
Kanyema et al.
force, it is not the sole influencer (Shi et al., 2013; Buttv et al.,
2014; Sun et al., 2020).
Viral genomes diverge from their prokaryotic and eukaryotic
counterparts due to their dependence on host cell machinery for
genome replication and protein synthesis. This co-evolution
impacts viral gene expression, protein synthesis, pathogenicity,
and immune evasion (Butt et al., 2014; Kumar et al., 2021).
Hence, a comprehensive exploration of CUB is necessary for
understanding viral evolution, adaptation, and genomic features.
CUB patterns serve as key indicators of genomic evolution (Chen
et al., 2014; He et al., 2019). Consequently, this study examined
the CUB of ASFV, with the findings expected to facilitate
understanding of viral strain evolutionary trends, elucidate
transmission pathways, and determine the speed of evolution.
Moreover, such knowledge is crucial for predicting gene
function, expression, regulation, and designing vaccines.
Materials and methods
Complete genome sequences of ASFV isolates/strains were
retrieved from the GenBank database via the web portal at
https://www.ncbi.nlm.nih.gov/data-hub/genome/. For an in-
depth understanding of ASFV codon usage bias, only viruses
with complete genomic information were considered in this
study. Following the rigorous elimination of redundancy and
potential recombination, 54 distinct strains (n = 54) from the
collected samples (n = 174) were selected and included in this
analysis (Supplementary Table S1). Ten Open Reading Frames
(ORFs) of each of the 54 retained strains were obtained from the
Open Reading Frame Finder available at https://www.ncbi.nlm.
nih.gov/orffinder/ for codon usage parameter estimation.
During the selection of sequences, the following criteria were
used; sequences with only ATG as a start codon were retrieved,
sequences with a minimal length of less than 75 were not
retrieved, and sequences with nested ORFs were also not
retrieved. For simplicity, the 10 longest ORFs, as one of the
options displayed in an ORF Finder, were selected. However,
sequences that had no terminating codons were removed and
replaced with the ones with stop codons.
Fundamental indices were computed using the online CAIcal
server available at http://genomes.urv.es/CAIcal/ (Puigbò et al.,
2008). These parameters include Relative Synonymous Codon
Usage (RSCU), Effective Number of Codons (ENC), Codon
Adaptation Index (CAI), and Relative Codon Deoptimization
Index (RCDI). Sus scrofa domestica’s coding sequences with
22094 codons used as a host reference genome, in this regard,
were retrieved from the Codon Usage Database available at
https://www.kazusa.or.jp/codon/ (Nakamura et al., 2000). The
Similarity Index (SiD) and Second Parity Rule Analysis, however,
were executed using the vhcub package in R software version 4.1.
3, which can be downloaded at https://cran.r-project.org/web/
packages/vhcub/ (Anwar et al., 2020). Hydropathicity and
Acta Virologica
10.3389/av.2023.11562
aromaticity indices were calculated using CodonW 1.4.
2 version available at http://codonw.sourceforge.net/. Principal
Component Analysis (PCA), which extracts major trends of
variations among the strains by transforming RSCU values
into uncorrelated variables, and the correlation analysis
between principal components and codon usage indices via
Spearman’s rank method were performed using R software.
The selected packages for PCA were factoextra and
FactoMineR, supported by ggplot2 (Husson et al., 2022;
Kassambara and Mundt, 2022; Wickham et al., 2022).
For phylogenetic analysis, sequence data was compiled and
edited using BioEdit (version 7.0.9.0). Multiple sequence
alignment (MAS) was executed online via MAFFT software
available at https://www.ebi.ac.uk/Tools/msa/mafft/, while the
phylogenetic tree was constructed using MEGA version 11,
downloadable at https://www.megasoftware.net/. The
subsequent section provides a detailed account of the codon
usage indices and other vital components.
Nucleotide composition
The nucleotide compositional constraints of the entire
genome ORFs were assessed using the CAIcal server
(http://genomes.urv.es/CAIcal/) (Puigbò et al., 2008). This
analysis involved the calculation of various measures
including the overall nucleotide frequency, the nucleotide
frequency at the third position of synonymous codons, the
average frequency of G and C nucleotides at the third codon
position (GC3), and the average frequency of G and C
nucleotides at the first and second codon positions(GC12).
The termination codons TGA, TAG, and TAA, as well as the
ATG codon for Methionine and the TGG codon for
Tryptophan, were excluded from this analysis.
Relative synonymous codon usage
Relative Synonymous Codon Usage (RSCU) refers to the
differential usage of synonymous codons that code for the same
amino acid. Essentially, RSCU is the ratio of the observed
frequency to the expected frequency of a codon, assuming
that all codons for a particular amino acid are used randomly
(Sharp and Li, 1986). The RSCU value is unaffected by amino
acid composition and is often used to estimate codon usage bias.
A higher RSCU value implies a higher frequency of codon usage
and a stronger codon usage bias. A codon is said to have a
positive codon usage bias if its RSCU value is >1.0, and a negative
codon usage bias if its RSCU value is <1.0. Codons with RSCU
values <0.6 are considered under-represented, whereas those
with values >1.6 are over-represented. Codons with RSCU
values of 1.0 are considered unbiased, and used at an equal
frequency (Sharp and Li, 1986).
Published by Frontiers
Institute of Virology
03 Biomedical Research Center, Slovak Academy of Sciences
Kanyema et al.
The following formula is used to calculate RSCU values for
each strain:
Xij
RSCUij
ni
1
ni

Xij
j
1
where Xij represents the occurrence frequency of the jth codon
for the ith amino acid (any Xij with a value of zero is at random
assigned a value of 0.5), and ni represents the number of codons
for the ith amino acid (ith codon family).
Principal component analysis
Principal component analysis (PCA) is a widely used
statistical method for analyzing CUB via a dimension
reduction strategy (Sharp and Li, 1986; Wold et al., 1987).
This analysis reveals the main trends among the correlated
variables (in this case, codons) across different ASFV strains.
The RSCU values for each strain are represented by a 59-
dimensional vector of codons. PCA then transforms these
RSCU values into uncorrelated variables captured by principal
components (Todorov et al., 2018). After this, the factors
influencing codon usage bias are assessed via correlation
analysis between the principal axes (PC1 & PC2) and codon
usage parameters.
Effective number of codons
The effective number of codons (ENC) provides a measure
of the extent to which codon usage in a gene or genome
deviates from equal use of synonymous codons (Wright,
1990). In ENC analysis, each codon is assigned an ENC
value. ENC values range from 20 to 61. Unlike RSCU, a
higher ENC value indicates less codon usage bias. Neither
the amino acid content nor the gene length impacts the
effective number of codons. ENC values less than or equal
to 45 generally signify strong codon usage bias (Comeron and
Aguade, 1998).
The standard ENC values were calculated using the formula:
9 1 5 3
ENC
2 + + + +
F2 F3 F4 F6
where; F (i = 2, 3, 4, 6) represents the mean Fi values for the i-fold
degenerate amino acids. The Fi value represents the likelihood
that two randomly chosen codons for an amino acid are identical.
Fi is determined as follows:
i
n
njn − 1
2 codon usage. To further investigate the influence of Sus scrofa
j
1
Fi

n−1
Acta Virologica
10.3389/av.2023.11562
where i is the type number of the synonymous codon family to
which the particular amino acid belongs, nj is the number of
observed counts of the jth codon for that amino acid, and n is the
total number of observed codon counts for the same amino acid.
Codon adaptation index
The Codon Adaptation Index (CAI) is a measure used to
quantify the similarity between the codon usage of viral genes or
genomes and their hosts (Sharp and Li, 1987). It is a widely used
method for assessing codon usage bias due to natural selection. It
indicates the adaptation of the virus to the host. The CAI value
ranges from 0 to 1, with higher values suggesting stronger host
adaptability. The CAI values were computed using the CAIcal
server available at http://genomes.urv.es/CAIcal/ to estimate the
ASFV codon adaptation to its host. The reference dataset of
synonymous codon usage was downloaded from the Codon
Usage Database available at https://www.kazusa.or.jp/codon/
(Nakamura et al., 2000).
Relative Codon Deoptimization Index
The Relative Codon Deoptimization Index (RCDI)
quantifies the similarity between a gene’s codon usage and
the codon usage of a reference genome (Mueller et al., 2006). It
can also be employed to assess the speed of viral gene
translation within a host genome and estimate the co-
evolution of the virus with its host. RCDI values near
1 indicate a virus that is highly adapted to its host and
follows the codon usage pattern of its host. RCDI values
greater than 1 suggest the virus is less well-adapted to the
host or has codon usage patterns different from its host (Butt
et al., 2016). The RCDI values were computed using the RCDI/
eRCDI server available at http://genomes.urv.es/CAIcal/
(Puigbò et al., 2010). The codon usage pattern for Sus
scrofa domesticus was retrieved from the Codon Usage
Database for use as reference, as was done for the CAI
calculation (Nakamura et al., 2000).
Similarity index analysis
The similarity index [SiD or D (A, B)] was utilized to estimate
the impact of overall host codon usage patterns on virus gene
expression (Zhou et al., 2013). SiD ranges between 0 and 1, with a
higher value indicating a stronger influence of the host on virus
codon usage patterns on ASFV codon usage, the similarity index
was computed as follows:
Published by Frontiers
Institute of Virology
04 Biomedical Research Center, Slovak Academy of Sciences
Kanyema et al.
59

ai bi
i
1
R(A, B)

59 59

ai2 ×
bi2
i
1 i
1
1 − R(A, B)
D(A, B)

2
where R(A, B) is defined as the cosine of the angle between
the spatial vectors A and B, representing the degree of
similarity between the overall usage patterns of the virus
and host codons. ai represents the RSCU value for a specific
codon from synonymous codons of the ASFV strain. bi
represents the RSCU value for the same codon in the host.
D (A, B) represents the potential impact of the host’s overall
codon usage on the ASFV strain. This value ranges from 0 to
1.0, with a higher value suggesting that the host has a strong
influence on codon usage.
Hydropathicity and aromaticity indices
The GRAVY score for hydropathicity and the Aroma
score for aromaticity were employed to estimate the
biochemical properties of translated proteins (Lobry and
Gautier, 1994). GRAVY stands for General Average
Hydropathicity; it ranges from −2 to 2, where negative and
positive values respectively indicate hydrophilic and
hydrophobic amino acids. The Aroma score indicates the
presence of aromatic amino acids such as tryptophan,
tyrosine, and phenylalanine in an encoded protein.
Hydropathy and aroma values provide insight into the
biochemical significance of the synthesized protein,
indicating the effects of natural selection on a viral
genome and, consequently, on the virus’s codon usage
(Chen et al., 2014; Yu et al., 2021b).
Neutral evolution analysis
In ASFV, neutral analysis was employed to assess the
changing influences of mutational pressure and natural
selection (Sueoka, 1988). This study presents the
GC12 values of the synonymous codons plotted against
the GC3 values. To analyze the evolutionary
characteristics of mutational pressure and natural
selection in ASFV, the GC12 or GC3 values are plotted
against evolutionary time. The slope of a simple regression
line signifies the evolutionary speed of mutational and
natural selection pressures. The influence of mutational
pressure and natural selection on base composition can be
assessed by examining the correlation between the GC
content at the first and second codon positions (GC12)
Acta Virologica
10.3389/av.2023.11562
and that at the third codon position (GC3). As a result,
the online software tool available at http://genomes.urv.es/
CAIcal/ was used to determine GC12 and GC3, and
regression analysis was then conducted using R. The
mutation-selection equilibrium coefficient, representing
the evolutionary speed of mutational pressure and natural
selection pressure, is the slope (regression coefficient) of the
regression line. If all points spread diagonally (slope = 1) and
the correlation between the GC3 and GC12 variables is
statistically significant, mutation is the primary factor
influencing codon usage. Otherwise, if the regression line
is parallel or inclined towards the horizontal axis (near-zero
slope), the selection is considered the dominant factor
(Sueoka, 1988).
ENC-GC3 plot analysis
ENC values were plotted against GC3 values in an ENC-
GC3 plot to discern the primary influences on codon usage
bias (Wright, 1990). In this diagram, the ENC value forms
the y-axis and the GC3 value constitutes the x-axis. Points
located on or slightly below the expected curve suggest
mutational pressure as the principal driver of codon
usage, whereas points considerably lower than the curve
indicate selection pressure as the dominant evolutionary
force. The ENC value for each GC3 is calculated as follows:
29
ENCexpected
2 + s +  2 
s + (1 − s)2
The s value represents the GC3s content of each codon.
Second parity rule
According to Chargaff’s second parity rule (PR2), the
mononucleotides A = T and G = C in the coding sequences
suggest that there is no bias for both natural selection and
mutational pressure (Sueoka, 1999a). The PR2 is plotted with
AT bias at the third codon position [A3/(A3+T3)] as ordinate
against GC bias at the third codon position [G3/(G3+C3)] as
abscissa to examine the influence of natural selection and
mutational pressure on the codon usage pattern. The origin
point (0.5, 0.5), at which the coordinates A = T and G = C are
located, indicates no bias for both natural selection and
mutational pressure. When the value is more than 0.5, it is
noticed that purine is preferred over pyrimidine (Sueoka,
1999b). The bias introduced by mutational pressure and
natural selection assists in determining the degree of
deviation from PR2. In other simple terms, a value greater
or less than 0.5 displays a bias caused either by natural
selection or mutational pressure or both.
Published by Frontiers
Institute of Virology
05 Biomedical Research Center, Slovak Academy of Sciences
Kanyema et al.
Dinucleotide frequency
The Emboss compseq tool available at https://www.
bioinformatics.nl/cgi-bin/emboss/compseq computed the
dinucleotide bias, which can affect codon usage in viruses and
other organisms. The bias is measured as the odds ratio (Pxy) of
observed over expected frequency (Karlin et al., 1994). Typically,
values above 1.23 suggest overrepresentation and values below 0.
78 suggest underrepresentation. The formula for calculation is:
fxy
Pxy

fxfy
where; fxy, f x, f y represent the frequency of dinucleotide xy, and
the frequency of nucleotide x, y respectively.
Phylogenetic analysis
After curating and editing all retrieved whole genome sequences
(n = 174) using Bioedit software, each preserving a size of 5500kb, they
were subjected to Multiple Sequence Alignment (MSA) via MAFFT
software available at https://www.ebi.ac.uk/Tools/msa/mafft.
Redundant strains were removed using Jalview software version 2.
11.2.5 available at https://www.jalview.org/download/windows/,
followed by the detection of putative recombination sites among
strains with the Recombination Detection Program (RDP4, version 4.
101) available at http://web.cbio.uct.ac.za/~darren/rdp.html. Putative
recombination sites in aligned sequences detected by at least
three different default methods (RDP, GENECONV,
CHIMAERA, MAXCHI, BOOTSCAN, SISCAN, 3SEQ)
were selected. A p-value less than 0.05 determined the
statistical significance. The retained sequences (n = 54)
underwent phylogenetic analysis after these meticulous
processes. The phylogenetic tree was estimated with MEGA
software version 11 using a Tamura-Nei model and the
Maximum Likelihood (ML) method. An initial tree was
inferred by maximum parsimony and tested for phylogeny
with 100 bootstraps. Other parameters remained at default
settings. The resulting tree, saved in NWK format, was
imported to iTOL (https://itol.embl.de/tree/) for annotation
and restructuring.
Software and statistical analysis
Spearman’s rank correlation was computed using R software
to identify significant factors influencing codon usage patterns. A
p-value of <0.01 was considered highly significant, while a
p-value of 0.01 < p < 0.05 indicated a significant correlation.
The Kruskal-Wallis test evaluated the significance between
groups in box plots at 0.05. Additionally, a correlogram was
generated using Past software version 4.03, available at https://
Acta Virologica
10.3389/av.2023.11562
past.en.lo4d.com/window. Based on the calculated RSCU values
of 59 codons for each ASFV strain/lineage, cluster analysis
(depicted as a heat map) was performed using the online tool
CIMminer6, available at https://discover.nci.nih.gov/cimminer/.
Results
Nucleobase composition
The overall nucleotide frequencies of ORFs, including A%, T
%, G%, and C%, were calculated, with results presented in
Supplementary Table S2. Other frequency calculations
included nucleotide at the third codon position (A3, T3, G3,
and C3), overall GC content frequency, average frequency of G +
C at the first and second codon positions (GC12), and average
frequency of G + C at the third codon position (GC3). A%
exhibited the highest overall nucleotide frequency (33.77 ± 0.86),
followed by T% (32.08 ± 0.80), with the lowest being C% (17.45 ±
1.01). However, G% (18.44 ± 0.52) displayed minimal variation
among the strains. The nucleotide frequency at the third codon
position mirrored the overall nucleotide content in a slightly
different pattern. The overall %GC content was 35.95 ± 1.35,
whereas the GC12 and GC3 contents were 35.96 ± 1.05 and
35.95 ± 2.10, respectively. Furthermore, GC showed a significant
correlation with GC12 and GC3, and these variables (GC12 &
GC3) also correlated with each other (Figure 12; Supplementary
Table S3). Nucleobase composition, GC content, and its
counterparts demonstrated a significant correlation with ENC,
highlighting their influence on codon usage bias. The prominent
role of GC content in codon usage indicates that mutational
pressure is the key factor.
Codon usage patterns
Relative Synonymous Codon Usage (RSCU) analysis
highlighted variation in codon usage among the strains
(Table 1), leading to the emergence of geographically distinct
lineages later confirmed by Principal Component Analysis
(PCA). Across all lineages, 26 codons exhibited positive bias
(with 5 overrepresented), and 26 codons showed negative bias
(with 9 underrepresented). The remaining 7 codons displayed
slightly uneven expression across lineages (indicated in green),
with 4 being negatively biased and 3 showing no apparent bias.
Interestingly, 22 of the 26 positively biased codons ended in
A or T (9 A-ended, 13 T-ended), indicating ASFV’s preference
for A/T-ended codons over G/C-ended ones. The five
overrepresented codons (RSCU>1.6) were all A/T-ended
(TTT, TTA, TCT, AGA, and GGA), while the nine
underrepresented codons (RSCU<0.6) were G/C-ended (TTC,
CTC, GTC, TCG, CCG, GCG, CAC, AAG, CGC).
Published by Frontiers
Institute of Virology
06 Biomedical Research Center, Slovak Academy of Sciences
Kanyema et al. 10.3389/av.2023.11562

TABLE 1 Mean RSCU values of each codon for the 59 codons in 54 strains (10 ORFs each) were averaged into lineages corresponding to their
geographical distribution. In addition, the column values for the host were included for comparison. A codon is said to have a positive codon
usage bias if its RSCU value is >1.0, and a negative codon usage bias if its RSCU value is <1.0. Codons with RSCU values <0.6 are considered under-
represented, whereas those with values >1.6 are over-represented. Codons with RSCU values of 1.0 are considered unbiased, and used at an equal
frequency. Different colors were used to signify codon usage variations: red=positive bias, overrepresented; blue=positive bias, represented;
straw=negative bias, underrepresented; black=negative bias, represented; green=average values of the codons expressed uneven across
lineages. When compared to the host, the lineages' codon usage appeared to be predominantly antagonistic, with just sporadic instances of
concordance. Principal component analysis eliminated codons that encodes for a single amino acid such as Methionine (Met) and Tryptophan
(Trp), as well as stop codons.

Lineage Host

Aminoacids Codons East African South African European Asian Avarage Sus scrofa domesticus

Phe TTT 1.66 1.63 1.62 1.63 1.64 0.97

Phe TTC 0.34 0.37 0.38 0.37 0.37 1.03
Leu TTA 1.71 1.68 1.66 1.78 1.71 1.46
Leu TTG 0.85 0.88 0.89 0.81 0.86 0.64
Leu CTT 1.3 1.3 1.3 1.32 1.31 0.62
Leu CTC 0.51 0.57 0.54 0.52 0.54 0.44
Leu CTA 0.9 0.82 0.85 0.87 0.86 2.5
Leu CTG 0.74 0.75 0.76 0.7 0.74 0.35
Ile ATT 1.3 1.28 1.27 1.29 1.29 1.3
Ile ATC 0.7 0.76 0.71 0.73 0.73 0.63
Ile ATA 1 0.96 1.02 0.98 0.99 1.07
Val GTT 1.1 1.18 1.14 1.13 1.14 0.56
Val GTC 0.5 0.56 0.49 0.56 0.53 0.43
Val GTA 1.29 1.3 1.33 1.23 1.29 1.93
Val GTG 1.11 0.96 1.04 1.08 1.05 0.41
Ser TCT 1.72 1.73 1.72 1.7 1.72 1.19
Ser TCC 1.34 1.47 1.39 1.26 1.37 0.93
Ser TCA 0.76 0.79 0.81 0.82 0.80 2.44
Ser TCG 0.34 0.24 0.27 0.32 0.29 0.21
Ser AGT 0.91 0.9 0.94 0.95 0.93 0.41
Ser AGC 0.92 0.87 0.87 0.95 0.90 0.81
Pro CCT 1.32 1.33 1.42 1.46 1.38 1.06
Pro CCC 1.14 1.06 1.08 1.05 1.08 1.39
Pro CCA 1 1.09 0.94 1.03 1.02 1.53
Pro CCG 0.53 0.51 0.55 0.47 0.52 0.02
Thr ACT 0.9 0.95 0.9 0.82 0.89 1.03
Thr ACC 1.21 1.21 1.24 1.25 1.23 0.87
Thr ACA 1.2 1.32 1.24 1.24 1.25 1.83
Thr ACG 0.69 0.52 0.62 0.69 0.63 0.27
Ala GCT 1.22 1.38 1.3 1.25 1.29 0.95
Ala GCC 1.19 1.02 1.17 1.26 1.16 0.91
Ala GCA 1.07 1.2 1.1 1.09 1.12 2.09
Ala GCG 0.52 0.4 0.43 0.4 0.44 0.05
Tyr TAT 1.43 1.32 1.36 1.38 1.37 0.93
Tyr TAC 0.57 0.68 0.64 0.62 0.63 1.07
His CAT 1.47 1.5 1.46 1.52 1.49 0.56
His CAC 0.53 0.5 0.54 0.48 0.51 1.1
Gln CAA 1.35 1.37 1.36 1.32 1.35 1.61
Gln CAG 0.65 0.63 0.64 0.68 0.65 0.07
Asn AAT 1.19 1.18 1.21 1.21 1.20 0.61
(Continued on following page)

Published by Frontiers
Institute of Virology
Acta Virologica 07 Biomedical Research Center, Slovak Academy of Sciences
Kanyema et al. 10.3389/av.2023.11562

TABLE 1 (Continued) Mean RSCU values of each codon for the 59 codons in 54 strains (10 ORFs each) were averaged into lineages corresponding to
their geographical distribution. In addition, the column values for the host were included for comparison. A codon is said to have a positive codon
usage bias if its RSCU value is >1.0, and a negative codon usage bias if its RSCU value is <1.0. Codons with RSCU values <0.6 are considered under-
represented, whereas those with values >1.6 are over-represented. Codons with RSCU values of 1.0 are considered unbiased, and used at an equal
frequency. Different colors were used to signify codon usage variations: red=positive bias, overrepresented; blue=positive bias, represented;
straw=negative bias, underrepresented; black=negative bias, represented; green=average values of the codons expressed uneven across lineages.
When compared to the host, the lineages' codon usage appeared to be predominantly antagonistic, with just sporadic instances of concordance.
Principal component analysis eliminated codons that encodes for a single amino acid such as Methionine (Met) and Tryptophan (Trp), as well as stop
codons.

Lineage Host

Aminoacids Codons East African South African European Asian Avarage Sus scrofa domesticus

Asn AAC 0.81 0.82 0.79 0.79 0.80 1.39

Lys AAA 1.42 1.41 1.41 1.41 1.41 1.43
Lys AAG 0.58 0.59 0.59 0.59 0.59 0.25
Asp GAT 1.33 1.38 1.29 1.31 1.33 1.24
Asp GAC 0.67 0.62 0.71 0.69 0.67 0.76
Glu GAA 1.35 1.38 1.38 1.39 1.38 1.76
Glu GAG 0.65 0.62 0.62 0.61 0.63 0.24
Cys TGT 1.37 1.39 1.38 1.43 1.39 0.74
Cys TGC 0.63 0.61 0.62 0.57 0.61 0.59
Arg CGT 1.06 1.07 1.04 1.09 1.07 1.32
Arg CGC 0.39 0.43 0.44 0.4 0.42 1.27
Arg CGA 1.03 0.97 0.91 0.85 0.94 3.41
Arg CGG 0.83 0.76 0.85 0.77 0.80 0
Arg AGA 1.82 1.89 1.85 1.96 1.88 0
Arg AGG 0.87 0.88 0.91 0.93 0.90 0
Gly GGT 0.85 0.88 0.92 0.96 0.90 0.71
Gly GGC 0.72 0.75 0.75 0.64 0.72 0.76
Gly GGA 1.65 1.75 1.68 1.71 1.70 1.24
Gly GGG 0.77 0.63 0.65 0.69 0.69 0.62

When compared to the host, codon usage appeared largely
antagonistic, with only sporadic instances of concordance (Table 1).
A heat map (Figure 1) was generated to reveal hidden patterns of
codon usage across lineages. Two distinct blocks were observed: a
green dominant block (primarily representing GC-ended codons,
except TCA, CGA and ACT) and a red dominant block (mainly
representing AT-ended codons, except GCC and CCC).
Upon close examination, it was evident that the green
dominant block was a negatively biased cluster of codons
(indicating less preferred codons) with blended patterns of
sporadic random codon usage. Conversely, the red dominant
block represented a positively biased cluster (indicating preferred
codons), also interspersed with patterns of random codon usage.
Principal Component Analysis
Principal Component Analysis (PCA) was conducted in R to
further reveal the patterns of relative synonymous codon usage.
The 59 codon dimensions, each having an RSCU value from
specific coding sequences, were condensed into principal
components. The first two components (PC1 = 36% and
PC2 = 16.9%) accounted for 52.9% of the total variation. The
following components, PC3, PC4, and PC5 retained 8.9%, 8.3%,
and 5.6% of the variation respectively.
A scree plot was generated to describe how much variation
each principal component retained from the original variables.
The first four PCs were sufficient to define the necessary
variation, but most of the analyses primarily focused on
PC1 and PC2 (Figure 2).
A variable factor map, in the form of a bar graph, was
prepared to summarize the top ten most influential variables
for each component. Paired component models were also
constructed to identify the contribution of two components.
Codons TGT, TGC, GAG, and CGC emerged as the most
influential in modeling PC1, while TCC, CTC, ACG, and
GCC greatly shaped PC2. Similar patterns were observed in
PC3, PC4, and in the 1–2, 2–3, and 3–4 component models
(Supplementary Figure S1).
A correlating variable plot was computed, revealing significant
patterns (Figure 3). On this plot, variables with similar patterns
(positive correlation) clustered together, such as those in the same

Published by Frontiers
Institute of Virology
Acta Virologica 08 Biomedical Research Center, Slovak Academy of Sciences
Kanyema et al. 10.3389/av.2023.11562

FIGURE 1
A Heat Map Illustrating RSCU Patterns Based on Lineages. The map delineates four distinct lineages, each represented by a separate column.
Rows primarily form two blocks: red for preferred A/T-ended codons and green for non-preferred G/C-ended codons, interspersed with sporadic
black patches indicating randomly chosen codons. The RSCU values are arranged into continuous ranges, resulting in observable color variations:
green for <1, black for 1–1.6, and red for >1.6. Cluster creation for the CIMminer heatmap analysis employed Euclidean distance and complete-
linkage methods.

Published by Frontiers
Institute of Virology
Acta Virologica 09 Biomedical Research Center, Slovak Academy of Sciences
Kanyema et al. 10.3389/av.2023.11562

FIGURE 2
A Scree Plot Indicating Variation Retained by Each Principal Component. The y-axis represents the proportion of explained variation, and the
x-axis represents the dimensions of the principal components. The first four components account for 75.7% of the necessary variation, but the
analysis primarily engaged the first two principal components, covering 52.9%.

FIGURE 3
A Variable Correlation Plot. It reveals two distinct clusters: an AT-ended cluster on the left and a GC-ended cluster on the right. Within each
cluster, members demonstrate a positive correlation, while members across clusters show a negative correlation. The strength of the correlation
between two members is gauged by their angle of inclusion.

Published by Frontiers
Institute of Virology
Acta Virologica 10 Biomedical Research Center, Slovak Academy of Sciences
Kanyema et al.
FIGURE 4
Continental-based Lineages and Their Corresponding Observations. The diagram showcases four lineages, each with observations sharing the
background color of the lineage.
quadrant. Conversely, variables grouped in opposite quadrants
demonstrated dissimilar patterns (negative correlation). The right
half of the circle revealed G/C-ended codons, while the left half
revealed A/T-ended codons. Additionally, the data indicated that
G/C-ended codons are negatively correlated to A/T-ended codons.
Four distinct groups were proposed based on geographical
region, namely: Europe, Asia, East Africa, and South Africa. With
the available data, we aimed to investigate whether these groups
exhibited unique codon usage patterns. Clear, albeit overlapping,
clusters emerged, indicating strains/isolates with similar patterns
grouping together (Figure 4). Data points were assigned
identification numbers for ease of reading. These numbers,
alongside detailed strain/isolate specifications, can be found in
Supplementary Table S1. For the Asian lineage, strains 48, 15, and
52 showed the highest codon usage patterns. Notably, number 32,
displayed to the right of PC1, exhibited extreme codon usage
characteristics. For the European lineage, the highest codon usage
patterns were observed in data points 40, 45, and 9. In contrast, the
East African lineage displayed the highest codon usage in data
points 2, 17, 18, 12, 30, 11, 41, 6, and 3, while the South African
lineage showed data points 22, 23, 24, 26, and 27 as the highest. All
data points that clustered near the origin in each lineage
represented average expression, including 37 and 14 for the
European lineage, 21 for the Asian lineage, 42 and 43 for the
East African lineage, and 5, 7, and 8 for the South African lineage.
Further, a biplot with loading vectors (codons) and individual
distributions was prepared to visualize the interrelationships
between different loadings and the individual observations they
Acta Virologica
10.3389/av.2023.11562
influence, as well as each loading’s correlation with a specific
principal component (PC). Clustering was utilized to discern these
variations in groups. In essence, an individual on the same side as a
given variable was deemed to have a high value for that variable,
while an individual on the opposite side of a variable was assumed
to have a low value for that variable. Positive loadings indicate a
positive correlation between a variable and a PC, whereas negative
loadings suggest a negative correlation. Variables covering longer
distances (more reddish) from the origin of a PC exert a significant
influence on their respective PCs and are well represented on the
factor map. Conversely, variables closer to the origin (more bluish)
have a lesser influence and are not well represented on that factor
map (Figure 5A).
For instance, variables such as CAG, TGC, CGC, CTG, CAC,
and GCG, due to their good representation on the factor map,
strongly influence PC1 (positive x-axis), while TGT, GAA, CAT,
TTA, GGT, and AGA significantly influence PC1 on the opposite
side. Similarly, ACG, TCG, GCC, GTG, AGC, TAT, and AGT,
due to their good representation on the factor map, exert more
effect on PC2 (positive y-axis) while TCC, CTC, TAC, ACA, and
GCA affect PC2 on the opposite side.
Less intense and shorter variables can also be well
represented on other PCs. For example, GAT, GAC, TTG,
CTA, and ACC variables are underrepresented on the Dim 1-
2 correlation circle but well-represented on the Dim 3-
4 correlation circle (Figure 5B).
Additionally, the smaller the angle between the variable and a
PC, the larger the correlation; conversely, a wider angle between
Published by Frontiers
Institute of Virology
11 Biomedical Research Center, Slovak Academy of Sciences
Kanyema et al. 10.3389/av.2023.11562

FIGURE 5
Codon Representation on the PC1 and PC2, and PC3 and PC4 Factor Maps. Arrows of varying lengths and color intensities denote the
continuous contribution of loading variables to the principal components.

Published by Frontiers
Institute of Virology
Acta Virologica 12 Biomedical Research Center, Slovak Academy of Sciences
Kanyema et al. 10.3389/av.2023.11562

FIGURE 6
Individual Distribution in Lineages Influenced by Loading Variables. Part A shows individuals and variables clustered together, with the color of
individual observations mirroring the background color of the cluster. A variable (codon) in line with an observation has a significant influence on that
observation, whereas a variable opposite an observation exerts minimal influence. Part B reveals clustered individuals without background color,
acting as a visibility-enhancing counterpart to Part A, particularly highlighting individual distribution in the absence of variables.

them signifies a smaller correlation and increased non-specificity.
With this in mind, all codons forming a positive angle with PC1
(positive x-axis) up to 45° strongly correlate positively with PC1; a
hypothesis that similarly applies to PC2 (positive y-axis). The
remaining codons in the same quadrants, including an angle from
45° to 90°, have a weak positive correlation with their respective
components, and these codons can also be redistributed to other
principal components. This hypothesis also applies to the codons
taking a negative angle from the origin on the opposite. In
summary, codons orienting near the horizontal to PC1 or
vertical to PC2 and close to the circumference are the most
influential to their respective PCs (as per the examples above).
From this analysis, it appears that a significant part of the
individual observations in the East African lineage expresses GC-

Published by Frontiers
Institute of Virology
Acta Virologica 13 Biomedical Research Center, Slovak Academy of Sciences
Kanyema et al.
ended codons (see clusters II and III on the positive PC1-axis and
cluster II on the positive PC2-axis, Figures 6A, B). Meanwhile,
a significant part of the Asian lineage observations appears to
express AT-ended codons (see cluster I on the positive PC2-
axis and negative PC1-axis). South African lineage
observations feature an expression of AT-ended codons on
both negative axes of PCs and GC-ended codons (mostly
C-ended codons) on the negative axis of PC2 (see cluster
IV for both negative directions of PCs and cluster III on the
negative direction of PC2). Finally, much like the East African
lineage, a significant part of the European lineage is greatly
influenced by a diversity of GC-ended codons (see cluster II
and III on the positive PC1-axis and cluster III on the negative
PC2-axis).
Codon usage bias and ENC-GC3 plot analysis
The Effective Number of Codons (ENC) is a prominent metric
for calculating Codon Usage Bias (CUB). The average ENC value for
all the coding sequences studied is 52.8 (range: 52.8–56.1 ± 0.99),
and the CUB, in this case, is generally low (greater than 45). Multiple
comparison box plots (Figure 7) revealed significant differences in
bias between lineages (p < 0.05).
The European lineage shows a slightly higher median value (as
indicated by the line within the box plot) than the East African
lineage, followed by the South African lineage in terms of magnitude.
However, the Asian lineage has the lowest median value of all.
Consequently, the European lineage demonstrates the least CUB,
FIGURE 7
Multiple Comparison Box Plots Depicting ENC. The codon usage bias is evaluated by comparing individual lineages. The box length signifies a
50% proportion of total data, with whiskers indicating the remaining 50%. An upper whisker represents 25% maximum values, and a lower whisker
depicts 25% minimum values. The box’s black line denotes the median, the red point represents the mean, and outliers are marked as black dots
beyond the whiskers.
Acta Virologica
10.3389/av.2023.11562
followed by the East African lineage, while the Asian lineage exhibits
the highest CUB.
Contrastingly, the East African lineage displays the most diversity
in its codon usage, followed by the European lineage, with the Asian
lineage being the least diverse. This diversity is apparent when
examining the range (R) and interquartile range (IQR), which
respectively measure the spread of the entire dataset and the
middle 50% of the data distribution. The East African lineage has
both the highest R and IQR, indicating its diverse codon usage. The
European lineage has a larger R than the South African and Asian
lineages but has a smaller IQR than these two lineages.
This apparent contradiction can be clarified by examining
the shape of the data distribution in addition to the data spread.
The East African lineage data is negatively skewed, meaning that
the lower portion of the box is longer than the upper portion and
that the median is closer to the upper end. Similarly, the
European lineage’s median is even closer to the upper end,
indicating a similar pattern. This pattern is not observed for
the South African and Asian lineages, which show a positive
skew, meaning the median aligns with or is positioned closer to
the lower end.
A negatively skewed distribution suggests the presence of
more low-score observations and fewer high-score observations.
The high-frequency data points are primarily represented in the
upper end, while the low-frequency data points are spread in the
lower end. Positively skewed data distribution shows the opposite
pattern.
These statistical insights suggest that the East African and
European lineages are dominated by consistently high ENC
Published by Frontiers
Institute of Virology
14 Biomedical Research Center, Slovak Academy of Sciences
Kanyema et al.
values, which biologically implies low CUB, allowing for more diverse
codon usage compared to the South African and Asian lineages.
Furthermore, independent of the distribution shape, the box of the
European lineage is above the South African and Asian lineages,
which can be interpreted as a high ENC score, and consequently, low
CUB. This allows a wider range of codon usage.
In conclusion, even though the interquartile range (IQR) of the
European lineage, as indicated by the length of the box, was less than
that of the South African and Asian lineages, the European lineage
still displayed greater codon usage diversity than the South African
and Asian lineages. The range (R) and the shape of the data
distribution, as statistical measures of group differences, provided
additional support for the European lineage, in contrast to the South
African and Asian lineages, which were only supported by IQR.
To better understand the primary evolutionary forces at
play—natural selection versus mutational pressure - in shaping
biased codon usage, we used the ENC-GC3 plot. Mutational
pressure is considered the sole factor when individual
observations lie on or just below the curve. However, when
observations fall significantly below the curve, it indicates that
other factors, such as natural selection, may also be contributing.
In this study, most observations were found to be well below the
curve (Figure 8), suggesting that natural selection may play a crucial
role in influencing codon usage bias.
Neutrality plot analysis
Further elucidation of the principal influences on codon usage was
achieved through neutrality plotting (Figure 9). The regression slope of
FIGURE 8
The ENC-GC3 Plot. The red line depicts the expected trend with data points distributed above, on, and below it. The significant underplacement
of points away from the curve suggests influences beyond mutational pressure, primarily natural selection, that shape the codon usage bias of ASFV.
Acta Virologica
10.3389/av.2023.11562
the GC12 versus GC3 plot nearing 0 suggests the preponderance of
natural selection, while a slope approximating 1 signifies the
dominance of mutational pressure, indicative of complete
neutrality. A robust correlation between GC12 and GC3 (R = 0.74,
p < 0.01) initially indicated the supremacy of mutational pressure over
natural selection. However, the regression equation’s slope, y =
0.43x+21, delineates the contributions of mutational pressure and
natural selection as 43% and 57% respectively, highlighting a slight
dominance of natural selection over mutational pressure.
The second parity rule
The construction of the parity graph assumed no bias towards
either mutational pressure or natural selection if the origin
coordinates were positioned at 0.5, 0.05 (Figure 10). In contrast,
the presence of bias, driven by either AT bias, GC bias, or both,
would instigate a departure from the PR2 rule where A≠T and
G≠C, and purines would be preferred over pyrimidines for
values >0.5. The mean values (0.62, 0.35) demonstrated a 1.2-
fold increase in AT bias and a 1.5-fold reduction in GC bias from
the origin. These findings underscore the predominance of natural
selection over mutational pressure, with both acting concurrently.
Protein biochemical features as determinants of
natural selection
The biochemical metrics (Gravy and Aroma scores)
assessed the impact of protein biochemistry on natural
Published by Frontiers
Institute of Virology
15 Biomedical Research Center, Slovak Academy of Sciences
Kanyema et al. 10.3389/av.2023.11562

FIGURE 9
A Neutrality Plot. This regression chart between GC12 and GC3 values discloses a 0.43 and 0.57 proportional contribution to evolution from
mutational and natural selection, respectively.

FIGURE 10
A Parity Plot. The y-axis represents an AT bias, and the x-axis denotes a GC bias. The proportional deviation of data points from the origin (0.5,
0.5) demonstrates a violation of the second parity rule. The AT bias deviates from the origin by an increase of 1.2, while the GC bias deviates by a
decrease of 1.5.

selection. Distinct lineage differences were clearly portrayed
through multiple comparison box plots (Figures 11A, B),
highlighting the role of hydropathicity and aromaticity in
codon usage bias. The divergence in these parameters across
lineages underscores the importance of natural selection in
shaping ASFV’s codon usage. Median gravy scores indicate
that African lineages, characterized by elongated boxes and
elevated medians, exceed Eurasian lineages in terms of
increased hydropathicity positivity. Similarly, African
lineages, with higher medians, showed a greater inclination
towards positive aromaticity compared to Eurasian lineages.
This trend suggests the geographical modulation of natural

Published by Frontiers
Institute of Virology
Acta Virologica 16 Biomedical Research Center, Slovak Academy of Sciences
Kanyema et al. 10.3389/av.2023.11562

FIGURE 11
Multiple Comparison Box Plots Displaying the GRAVY and Aroma Scores. The scores are assessed by juxtaposing individual lineages. The box
length denotes a 50% proportion of total data, with the whiskers illustrating the remaining 50%. An upper whisker represents 25% maximum values,
and a lower whisker shows 25% minimum values. The box’s black line indicates the median, the red point shows the mean, and outliers are
represented as black dots beyond the whiskers.

selection, manifesting as continental differences at the protein
biochemistry level. Subsequent correlation analysis of the
gravy and aroma scores with the first two principal
components confirmed a significant correlation, further
substantiating the influence of natural selection on codon
usage.
Codon Adaptation Index (CAI)
The Codon Adaptation Index (CAI) was employed to evaluate
the adaptation of ASFV genomes to their respective hosts, gauging the
influence of natural selection on viral genomic expression. A higher
CAI value (0–1) corresponds to greater adaptation. The expected

Published by Frontiers
Institute of Virology
Acta Virologica 17 Biomedical Research Center, Slovak Academy of Sciences
Kanyema et al.
value (eCAI) calculated from the coding sequences yielded 0.598 (p <
0.05), indicating that 60% of the viral codon usage is adaptive.
Although ASFV demonstrates real codon adaptation, it manifests
as moderate codon expression, with 40% of viral codons unutilized in
active replication cycles, potentially expressed during latency phases
(Kumar et al., 2018). Correlation analysis with PC1 and PC2, akin to
other codon usage parameters, revealed a significant correlation,
verifying ASFV adaptation. Furthermore, a correlation analysis
between CAI and ENC was conducted to validate this adaptation
as a result of natural selection’s influence on CUB.
Relative Codon Deoptimization Index
The RCDI established codon usage similarity at the gene level.
Unlike CAI, a higher RCDI value (>1) signifies codon use
dissimilarity (codon deoptimization and reduced adaptability)
relative to the host. Conversely, an RCDI value close to
1 indicates codon use similarity (codon optimization and viral
adaptability). The expected RCDI value derived from this study
was 3.003 (p < 0.05), denoting that the virus’s codon expression
diverges from the host’s, implying a low to moderate level of
adaptation. In other words, ASFV chooses not to express certain
codons during gene expression, thus surrendering that codon usage
gap to the host. This codon deoptimization strategy avoids host
competition and somewhat restricts viral translation speed, thereby
controlling viral replication rate (Butt et al., 2014; Butt et al., 2016).
This offers a unique advantage by moderating translation speed to
FIGURE 12
Correlogram Illustrating Autocorrelation Among Variables. The cycle’s relative size represents the correlation strength, mirrored by various
color intensities. Both positive and negative correlations are depicted continuously, ranging from a maximum (+1) for a strong positive correlation to
a minimum (−1) for a strong negative correlation. Areas without cycles suggest the absence of correlation between the variables.
Acta Virologica
10.3389/av.2023.11562
ensure optimal protein folding for effective replication cycles. The
virus may selectively utilize codons that the host does not prefer for its
survival, fitness, and immune evasion (Butt et al., 2014; Butt et al.,
2016; Yao et al., 2020).
Similarity index analysis
Beyond the aforementioned adaptation indices, we sought to
ascertain the influence of the host on overall codon usage in viral
gene expression. The Similarity Index (SiD) provided this
perspective. A higher value, nearing 1 (range, 0–1), signifies a
strong overall impact of the host on viral codon usage patterns.
The host impacted ASFV codon usage patterns by approximately
50% (0.49), implying that around half of the viral codons in any
gene expression remain unaffected by the host.
Correlation analysis
Autocorrelation was conducted to ascertain the
association among nucleotide base composition parameters,
ENC, CAI, GRAVY, and Aroma scores, and the first two
principal components. The aim was to identify a significant
relationship between these indices and PC1 and PC2.
Additionally, the length of the open reading frame (ORF)
was considered to validate the hypothesis that gene length
influences codon usage patterns. The first two components
Published by Frontiers
Institute of Virology
18 Biomedical Research Center, Slovak Academy of Sciences
Kanyema et al. 10.3389/av.2023.11562

were chosen as they encapsulated a substantial proportion of
CUB (52.9%). The results (Supplementary Table S3;
Figure 12) portrayed varied correlation patterns among
parameters at different significance levels (0.001<p< 0.05).
The correlogram showed a significant correlation between the
overall nucleotide base composition and the nucleotide base at
the third codon position with either PC1 or PC2 (0.001<p<
0.05), implying that nucleotide compositional constraint is a
determinant of CUB. A highly significant positive correlation
(p < 0.01) was observed between ENC and PC1, with a similar
positive correlation noted between CAI and PC1. Conversely,
the Gravy and Aroma scores displayed a significant correlation
(p < 0.05) with PC2. The length of the coding sequences was
positively correlated with both PC1 and PC2, highlighting the
importance of gene length in CUB. Additionally, a variable
correlation plot was drafted to examine the correlation between
loading vectors, in this case, codons (Figure 3).
ACG, GCG, CGT, CGC, CGA, and CGC) from the earlier
RSCU values were reassessed. Four of these (TCG, CCG, GCG,
CGC) were underrepresented, verifying the presence of
dinucleotide usage bias. Interestingly, the dinucleotide TρA,
usually found to be underrepresented in many studies, was
slightly overrepresented in this research (ρxy = 1.356) (Cheng
et al., 2013). This was supported by the presence of TTA among
the five overrepresented codons (TTT, TTA, TCT, AGA, and
GGA) in the RSCU values. Furthermore, an influence from the
positive AT bias in nucleotide composition contributed to the
overrepresentation of the dinucleotide TρA in ASFV. The
dinucleotide TT was overrepresented in the RSCU values of
the TTT and TTA codons, whereas the dinucleotides AA and
AT were not overrepresented.
Phylogenetic analysis

The evolutionary relationships of ASFV’s coding sequences,

Dinucleotide frequency analysis
The dinucleotide frequency analysis estimated the relative
abundance of 16 dinucleotides in ASFV’s coding sequences.
None of the dinucleotides were at their expected frequency or
randomly distributed (Table 2). The results indicated an over-
representation of dinucleotides AA, TT, AT, and TA
(ρxy >1.23), whereas underrepresented dinucleotides
included CG, CC, GC, GG, and GT (ρxy<0.78). To confirm
this, eight codons featuring the dinucleotide CpG (TCG, CCG,
based on geographical lineage, were reaffirmed through the
construction of a phylogenetic tree (Figure 13A). The lineages
were grouped into Asian, European, East African, and South
African clades. Interestingly, however, some strains from
different clades were found nested within foreign clades; this
could be partly attributed to genotypic relationships between
some of the strains and the parent clades where they originate.
This observation is supported by a phylogenetic tree
constructed based on genotypes, where nesting was
eradicated except for the French strain (MN913970.1/strain

TABLE 2 None of the dinucleotides were distributed randomly or at their predicted frequency. Values above 1.23 (ρxy >1.23) and below 0.78
(ρxy <0.78) indicate overrepresentation and underrepresentation, respectively. The variance in dincucleotide usage is indicated by colors. Black =
represented, Blue = underrepresented, and Red = overrepresented. STDs are denoted by maroon.

Dinucleotide East African South African European Asian Average STD

AA 1.998 1.857 2.003 2.041 1.974 0.08

AC 0.834 0.860 0.856 0.837 0.847 0.01
AG 0.886 0.849 0.902 0.911 0.887 0.03
AT 1.656 1.688 1.593 1.659 1.649 0.04
CA 0.946 1.038 0.981 0.989 0.989 0.04
CC 0.587 0.620 0.617 0.584 0.602 0.02
CG 0.385 0.344 0.390 0.347 0.367 0.02
CT 0.898 0.917 0.892 0.874 0.895 0.02
GA 1.049 0.971 1.066 1.062 1.037 0.04
GC 0.613 0.592 0.621 0.580 0.602 0.02
GG 0.660 0.611 0.694 0.647 0.653 0.03
GT 0.664 0.690 0.664 0.660 0.670 0.01
TA 1.380 1.386 1.303 1.354 1.356 0.04
TC 0.782 0.847 0.785 0.793 0.802 0.03
TG 1.056 1.061 1.062 1.046 1.056 0.01
TT 1.606 1.669 1.571 1.613 1.615 0.04

Published by Frontiers
Institute of Virology
Acta Virologica 19 Biomedical Research Center, Slovak Academy of Sciences
Kanyema et al. 10.3389/av.2023.11562

FIGURE 13
A Maximum Likelihood (ML) Phylogeny. The tree was constructed using the Tamura-Nei model and processed with 100 bootstraps in MEGA 11 software
for about 5 hours. The tree, saved in NWK format, was imported to iTOL online software for annotation. Isolates with analogous characteristics are grouped
into a single clade, while those with distinct features form different clades. Tree A reveals the evolutionary relationships of the continental lineages, with some
isolates nested in foreign clades, identified by color differentiation from the background. Tree B demonstrates the evolutionary relationships of the
genotypic lineages, with most nested isolates from Tree A absent, indicating a genotypic relationship independent of geographic distribution.

Liv13/33 (OmLF2) and South African strain (AY261362.1/ members. All these observations elucidate the complex
isolate Mkuzi 1979) (Figure 13B). These strains were found transmission chains and evolutionary dynamics of ASFV in
nested in the genotype II clade even though they are genotype I nature.

Published by Frontiers
Institute of Virology
Acta Virologica 20 Biomedical Research Center, Slovak Academy of Sciences
Kanyema et al.
Discussion
The nucleotide and codon usage composition showed a
significant correlation with ENC, indicating its influence on
CUB (Ma et al., 2017; Gu et al., 2020). The GC content (35.95%)
characterizes ASFV as an AT-rich DNA virus. ASFV’s preference for
using AT nucleotides over GC nucleotides in all its coding sequences
has been observed in a comparative genomics analysis of ASFV (Pu
et al., 2023). Other viruses with a similar AT nucleotide composition
include Hantaan viruses (Ata et al., 2021), Hepadnaviridae (Deb et al.,
2020), Flaviviridae (Yao et al., 2019), lyssaviruses (Zhang et al., 2018),
PEDV (Chen et al., 2014), PDCoV (He et al., 2019), and SARS-CoV-2
(Hou, 2020).
The significant correlation of GC content with ENC suggests that
mutational pressure is the evolutionary force driving the observed
biased codon usage (Zhou et al., 2013; Deb et al., 2021b). However, the
weak to moderate correlation of nucleotide content and codon
composition with the first two principal axes suggests that other
factors, such as natural selection, are also in play alongside mutational
pressure.
The ENC, an absolute measure of CUB, was 52.80, indicating
that ASFV has a low codon usage bias (>45) (Hemert et al., 2016).
This result aligns with those for PDCoV (ENC = 52.69) (Peng
et al., 2022), SARS-CoV-2 (ENC = 45.38) (Hou, 2020), PEDV
(ENC = 47.91) (Chen et al., 2014), MERS-CoV (ENC = 49.82)
(Chen et al., 2017), and other viruses.
The low biased codon usage value suggests that ASFV uses a wide
range of its synonymous codons within a respective codon family for
efficient translation and subsequent productive viral replication cycles
(Hemert et al., 2016). The significant difference in lineages’ENC
shown by box plots (Figure 7) supports the hypothesis that natural
selection under geographical or environmental influence, in addition
to mutational pressure, accounts for the observed viral evolutionary
differences. Furthermore, ENC-GC3, PR2, and neutrality plots
demonstrated that both natural selection and mutational pressure
are major drivers for evolution in ASFV. However, the neutrality plot
analysis revealed that natural selection is somewhat more influential
than mutational pressure by 14%.
Codon usage patterns, as indicated by RSCU, revealed 22 out of
26 positively biased codons (RSCU>1) as A/T-ended. Unsurprisingly,
all five overrepresented codons (RSCU>1.6) were also A/T-ended.
Conversely, all nine underrepresented codons (RSCU<0.6) were
G/C-ended. RSCU values also uncovered lineage-based codon
usage patterns, a finding further substantiated by PCA. The
striking contrast between the codon usage expression of the viral
lineages and that of the host suggests that ASFV experiences lower
translation efficiency, thereby allowing proteins to fold properly
during translation (Table 1). These findings suggest that natural
selection has a greater impact than mutational pressure.
The heat map representation of RSCU lineage patterns
(Figure 1) presented two distinct codon blocks: an A/T-ended
block and a G/C-ended block. It was observed that the A/T-
ended block was highly expressed (RSCU>1), while the G/C-
Acta Virologica
10.3389/av.2023.11562
ended block was least expressed (RSCU<1), which reiterates the
dominance of A/T-ended codons in ASFV.
PCA identified four clusters, each corresponding to
geographically-oriented lineages with individual observations.
However, these clusters overlap one another suggesting that
mutational pressure is a common evolutionary force at play
among strains/isolates, regardless of their geographical origins.
High codon usage patterns within clusters signal strains/isolates
with pronounced CUB in each region. Interestingly, lineages
were seen to favor GC-end, AT-end, or both, in their codon
usage. For instance, the East African and European lineages
skewed heavily toward G/C-ended codons, while the Asian
lineage favored A/T-ended codons. Only the South African
lineage seemed to be influenced by both codon ends. East
African lineage displayed the most diverse codon usage,
closely followed by the European lineage, as indicated by the
detailed CUB analysis using ENC and supported by PCA.
Conversely, the Asian lineage appeared to show the least
codon usage diversity. These findings suggest that, in addition
to mutational pressure, natural selection likely plays a significant
role in ASFV evolution.
GRAVY and Aroma scores indicated significant differences
among geographically-based lineages (p < 0.05). These scores
further support the influence of natural selection as a
fundamental force in ASFV evolution (Chen et al., 2014; Yu
et al., 2021b). This was especially apparent when multiple
comparison box plots showed the divergence of African and
Eurasian lineages in terms of their hydropathicity and
aromaticity tendencies. As indicators of natural selection,
these scores also positively correlated with PC2, providing
additional evidence that natural selection slightly exceeds
mutational pressure.
Indicators of adaptation such as the Codon Adaptation Index
(CAI, eCAI = 0.598), Relative Codon Deoptimization Index
(RCDI, 3.003>1), and Similarity Index (SiD, 0.49) all support
the notion that ASFV is co-adapting with its host in some way.
These metrics demonstrate that ASFV has moderately adaptive
features and has managed to deoptimize its codons to some
extent. Notably, the Similarity Index indicates that ASFV prefers
to express 50% of its codons to reduce competition with the host
during active replication cycles (Butt et al., 2014; Butt et al.,
2016). This antagonistic codon usage, also observed in hepatitis A
virus, shrimp viruses, and chikungunya virus (Andrea et al., 2011;
Butt et al., 2014; Tyagi et al., 2017), suggests that ASFV is not
highly host-specific, given its ability to infect hosts other than
domestic pigs, such as warthogs that serve as reservoirs for
maintaining sylvatic cycles (Lubisi et al., 2005; Costard et al.,
2009; Brown and Bevins, 2018; Tian et al., 2018; Craig et al.,
2021). Additionally, the CAI of 60% supports the recurrent
assertion of natural selection’s influence (Kumar et al., 2018).
The marked decrease in CpG dinucleotides may be a
response to host antiviral defenses triggered by unmethylated
CpG, which is recognized as a pathogenic signature by the host
Published by Frontiers
Institute of Virology
21 Biomedical Research Center, Slovak Academy of Sciences
Kanyema et al.
(Wang et al., 2016). To evade the host’s immune responses,
ASFV may reduce its CpG expression (Tyagi et al., 2017;
Mordstein et al., 2021). Another potential reason is the high
stack energy of CpG, which could impede translation and
replication in DNA duplexes. The lack of overrepresentation
of TpG and CpA dinucleotides suggests that methylation and
subsequent deamination of cytosine to thymidine is not a major
factor, consistent with several studies (Shackelton et al., 2006;
Cheng et al., 2013). Interestingly, the slight overrepresentation of
TpA similar to the Nipah virus was contrary to many organisms
that avoid TpA-containing codons to prevent nonsense
mutations (Giallonardo et al., 2017; Khandia et al., 2019;
Mordstein et al., 2021).
The constructed phylogenetic tree illustrates the evolutionary
relationships between lineages and how natural selection,
influenced by geographical factors, shapes ASFV evolution.
Some strains nested in foreign clades suggest not only the
influence of natural selection but also the potential impact of
mutational pressure at the codon level, leading to genotypically
similar clades. This inference is supported when a genotype-
based tree is constructed; wherein these nested strains no longer
appear, reflecting their genotypic relationship with the foreign
clades.
Conclusion
African Swine Fever (ASF) is a devastating disease that
severely impacts porcine populations, with substantial socio-
economic consequences. Therefore, studying the evolutionary
biology of the ASF virus (ASFV) is crucial for understanding its
replication cycle, complex transmission chain, and adaptation
mechanisms. Such understanding can also significantly aid in the
development of effective vaccines and therapeutics.
In this study, we conducted a comprehensive exploration of
the codon usage bias (CUB) and viral adaptation of ASFV. Our
analysis revealed a delicate balance between two major
evolutionary forces: natural selection and mutation pressure,
which mutually influence the evolution of ASFV. While the
viral lineages were distinct and primarily continent-based,
they showed a degree of interconnectedness.
Although both natural selection and mutational pressure
were found to contribute to ASFV evolution, natural selection
appeared to be slightly more predominant. This may be due to
the fact that while all viruses experience rapid mutation, ASFV,
being a large DNA virus, may not mutate as quickly as RNA
viruses. Future live experiments and evolutionary studies are
recommended to further investigate this hypothesis.
To our knowledge, this is the first comprehensive analysis of
codon usage for the entire ASFV genomic sequences. This work
could be valuable for studies investigating viral gene expression
and regulation, gene function prediction, parasite-host interaction,
and immune dysfunction, and for the design of drugs and vaccines.
Acta Virologica
10.3389/av.2023.11562
Data availability statement
The original contributions presented in the study are
included in the article/Supplementary Material, further
inquiries can be directed to the corresponding authors.
Author contributions
Research idea and design: MK, MC, XC, CW; Resource
mobilization and funding: XC, CW; Sample collection and
curation: MK, MC, JL, ML; Data analysis, collection and
interpretation: MK, MC, XX, YS, JW, YL; Writing and
editing: MK, MC, CS, YZ, GY; Supervision and knowledge
contribution: XC and CW. The article’s contribution was
reviewed and approved by all authors prior to submission.
Funding
This work was supported by the National Natural Science
Foundation of China (U21A20261, 32273043, 32202890,
31941018), the Science and Technology Development
Program of Jilin Province (20200402041NC), the Science and
Technology Development Program of Changchun City (21ZY42)
and China Agriculture Research System of MOF and MARA
(CARS-35).
Conflict of interest
The authors declare that the research was conducted in the
absence of any commercial or financial relationships that could
be construed as a potential conflict of interest.
Acknowledgments
The authors acknowledge the Chinese Scholarship Council
(CSC) for the scholarship facilitation of a PhD student who is
among the primary authors of this work.
Supplementary material
The Supplementary Material for this article can be found
online at: https://www.frontierspartnerships.org/articles/10.3389/
av.2023.11562/full#supplementary-material
SUPPLEMENTARY FIGURE S1
Codons most influential in PC1, PC2, PC3, PC1 and PC2, PC2 and PC3,
and PC3 and PC4. The first 10 codons that contributed the most are
listed on the X-axis, and the Y-axis displays the percentage
contribution. TGT, TGC, GAA, GAG, CGC, CAT, CAC, AGA, CTG, and GCG
are the codons identified for PC1. TCC, CTC, ACG, GCC, TAT, TAC, ACA,
Published by Frontiers
Institute of Virology
22 Biomedical Research Center, Slovak Academy of Sciences
Kanyema et al.
GCA, ATC, and TCG are the codons identified for PC2. CTA, ACC, GTG,
CCA, TTG, GAC, GAT, TCA, TTC, and TTT are the codons identified for
PC3. Codons TGT, TGC, CGC, GAA, GAG, CTC, TCC, ACG, CTG, and TTA
have been identified for the PC1 and PC2 combined model. Codons
TCC, CTA, ACC, CTC, GTG, ACG, ACA, GCC, CGT, and GCA have been
identified for the PC2 and PC3 combined model. Codons CAC, GAT,
CTA, ACC, TTG, ACT, CCA, CGA, CCT, and CTT have been identified for
the PC3 and PC4 combined model.
SUPPLEMENTARY TABLE 1
Data indicating isolates/strains and ORFs. Isolate information is provided
on Sheet 1 and ORF information is provided on Sheet 2. Sheet 1 has six
columns; including one for the accession numbers of the isolates, one
for the type of isolates, one for the genotypes of isolates, one for the
country of origin of the isolates, one for the year of isolation, and one for
the Principal Component Analysis (PCA) Identity Number (ID) for ease
of reading as indicated on the identified factor map.
References
Alkhamis, M. A., Gallardo, C., Jurado, C., Soler, A., Sa, M., and Arias, M. (2018).
Phylodynamics and evolutionary epidemiology of African swine fever p72-CVR genes in
Eurasia and Africa. PLoS ONE 13 (2), 01925655–e192618. doi:10.1371/journal.pone.0192565
Andrea, L. D., Pintó, R. M., Bosch, A., Musto, H., and Cristina, J. (2011). A
detailed comparative analysis on the overall codon usage patterns in hepatitis a
virus. Virus Res. 157, 19–24. doi:10.1016/j.virusres.2011.01.012
Anwar, A. M., Soudy, M., and Mohamed, R. (2020). “vhcub: virus-host codon
usage co-adaptation analysis [version 1; peer review:2 approved],” in F1000Res. 8
(2137), 1–10. Available at: https://doi.org/org/10.12688/f1000research.21763.1.
Ata, E. B., Li, Z. J., Shi, C. W., Yang, G. L., Yang, W. T., and Wang, C. F. (2022).
African swine fever virus: a raised global upsurge and a continuous threaten to pig
husbandry. Microb. Pathog. 167, 105561. doi:10.1016/J.MICPATH.2022.105561
Ata, G., Wang, H., Bai, H., Yao, X., and Tao, S. (2021). Edging on mutational bias,
induced natural selection from host and natural reservoirs predominates codon usage
evolution in hantaan virus. Front. Microbiol. 12, 699788–699818. doi:10.3389/fmicb.
2021.699788
Ayanwale, A., Trapp, S., Guabiraba, R., Caballero, I., and Roesch, F. (2022). New
insights in the interplay between african swine fever virus and innate immunity and
its impact on viral pathogenicity. Front. Microbiol. 13, 958307–958309. doi:10.3389/
fmicb.2022.958307
Bera, B. C., Virmani, N., Kumar, N., Anand, T., Pavulraj, S., Rash, A., et al. (2017).
Genetic and codon usage bias analyses of polymerase genes of equine influenza virus and
its relation to evolution. BMC Genomics 18, 652–718. doi:10.1186/s12864-017-4063-1
Brown, V. R., and Bevins, S. N. (2018). A review of african swine fever and the potential
for introduction into the United States and the possibility of subsequent establishment in
feral swine and native ticks. Front. Veterinary Sci. 5, 11–18. doi:10.3389/fvets.2018.00011
Butt, A. M., Nasrullah, I., Qamar, R., and Tong, Y. (2016). Evolution of codon
usage in zika virus genomes is host and vector specific. Emerg. Microbes Infect. 5,
1–14. doi:10.1038/emi.2016.106
Butt, A. M., Nasrullah, I., and Tong, Y. (2014). Genome-wide analysis of codon
usage and influencing factors in chikungunya viruses. PLoS ONE 9 (3),
e90905–e90920. doi:10.1371/journal.pone.0090905
Chen, S. N., Li, C. L., Lin, J. S., Zhai, S. L., and Sun, M. F. (2022). Diverse african
swine fever viruses in China. New Microbes New Infect. 46, 100976. doi:10.1016/j.
nmni.2022.100976
Chen, Y., Shi, Y., Deng, H., Gu, T., Xu, J., Ou, J., et al. (2014). Characterization of
the porcine epidemic diarrhea virus codon usage bias. Infect. Genet. Evol. 28,
95–100. doi:10.1016/j.meegid.2014.09.004
Chen, Y., Xu, Q., Yuan, X., Li, X., Zhu, T., Ma, Y., et al. (2017). Analysis of the
codon usage pattern in middle East respiratory syndrome coronavirus. Oncotarget 8
(66), 110337–110349. doi:10.18632/oncotarget.22738
Cheng, X., Virk, N., Chen, W., Ji, S., Ji, S., Sun, Y., et al. (2013). CpG usage in RNA
viruses: data and hypotheses. PLoS ONE 8 (9), e74109–9. doi:10.1371/journal.pone.
0074109
Comeron, J. M., and Aguade, M. (1998). An evaluation of measures of
synonymous codon usage bias. J. Mol. Evol. 47, 268–274. doi:10.1007/pl00006384
Costard, S., Wieland, B., Glanville, W. D., Jori, F., Rowlands, R., Vosloo, W., et al.
(2009). African swine fever: how can global spread be prevented. Philosophical
Trans. R. Soc. B 364, 2683–2696. doi:10.1098/rstb.2009.0098
Acta Virologica
10.3389/av.2023.11562
SUPPLEMENTARY TABLE 2
Proportions of nucleotides and codons at different positions. %A, %C, %T
and %G indicate proportion of the overall nucleotide composition of
Adenine, Cytosine, Thiamine and Guanine respectively whereas %A3,
%C3, %T3 and %G3 indicate proportion of the respective nucleotides at
third nucleotide position. %GC indicate overall proportion of GC content
whereas %GC3 indicate proportion of GC content at third codon position
and %GC12 indicate proportion of GC content at first and second codon
position. Effective Number of Codons (ENCs) has been indicated to
appreciate the absolute codon usage bias at strain/isolate level.
SUPPLEMENTARY TABLE 3
Autocorrelation illustrating correlation between variables. Positive and
negative correlations are depicted in numbers with continuous values:
p <0.01 indicates very significant correlation while 0.01 < p < 0.05
indicates significant correlation. Zero values indicate absence of
correlation.
Craig, A. F., Schade-Weskott, M. L., Harris, H. J., Heath, L., Kriel, G. J. P.,
Schalkwyk, L. V., et al. (2021). Extension of sylvatic circulation of african swine fever
virus in extralimital warthogs in South Africa. Front. Veterinary Sci. 8,
746129–746211. doi:10.3389/fvets.2021.746129
Deb, B., Uddin, A., and Chakraborty, S. (2020). Codon usage pattern and its
influencing factors in different genomes of hepadnaviruses. Archives Virology 165,
557–570. doi:10.1007/s00705-020-04533-6
Deb, B., Uddin, A., and Chakraborty, S. (2021a). Composition, codon usage
pattern, protein properties, and influencing factors in the genomes of members of
the family anelloviridae. Archives Virology 166 (0123456789), 461–474. doi:10.
1007/s00705-020-04890-2
Deb, B., Uddin, A., and Chakraborty, S. (2021b). Genome-wide analysis of codon
usage pattern in herpesviruses and its relation to evolution. Virus Res. 292, 198248.
doi:10.1016/j.virusres.2020.198248
Dixon, L. K., Chapman, D. A. G., Netherton, C. L., and Upton, C. (2012). African
swine fever virus replication and genomics. Virus Res. 173 (1), 3–14. doi:10.1016/j.
virusres.2012.10.020
Giallonardo, F. D., Schlub, T. E., Shi, M., and Holmes, E. C. (2017). Dinucleotide
composition in animal RNA viruses is shaped more by virus family than by host
species. J. Virology 91 (8), 1–15. doi:10.1128/JVI.02381-16
Gu, H., Chu, D. K. W., Peiris, M., and Poon, L. L. M. (2020). Multivariate analyses
of codon usage of SARS-CoV-2 and other betacoronaviruses. Virus Evol. 6 (1),
veaa032–10. doi:10.1093/ve/veaa032
He, W., Wang, N., Tan, J., Wang, R., Yang, Y., Li, G., et al. (2019). Comprehensive
codon usage analysis of porcine deltacoronavirus. Mol. Phylogenetics Evol. 141,
106618–106710. doi:10.1016/j.ympev.2019.106618
Hemert, F. V., van der Kuyl, A. C., and Berkhout, B. (2016). Impact of the biased
nucleotide composition of viral RNA genomes on RNA structure and codon usage.
J. General Virology 97, 2608–2619. doi:10.1099/jgv.0.000579
Hou, W. (2020). Characterization of codon usage pattern in SARS-CoV-2.
Virology J. 17, 138–210. doi:10.1186/s12985-020-01395-x
Husson, F., Josse, J., Le, S., and Mazet, J. (2022). FactoMineR: multivariate exploratory
data analysis and data mining, the comprehensive R archive network. Available at:
https://cran.r-project.org/package=FactoMineR (Accessed December 20, 2022).
Iyer, L. M., Aravind, L., and Koonin, E. V. (2001). Common origin of four diverse
families of large eukaryotic DNA viruses. J. Virology 75 (23), 11720–11734. doi:10.
1128/jvi.75.23.11720-11734.2001
Jia, N., Ou, Y., Pejsak, Z., Zhang, Y., and Zhang, J. (2017). Roles of African swine
fever virus structural proteins in viral infection. J. Veterinary Res. 61, 135–143.
doi:10.1515/jvetres-2017-0017
Karlin, S., Doerfler, W., and Cardon, L. R. (1994). Why is CpG suppressed in the
genomes of virtually all small eukaryotic viruses but not in those of large eukaryotic
viruses? J. Virology 68 (5), 2889–2897. doi:10.1128/jvi.68.5.2889-2897.1994
Kassambara, A., and Mundt, F. (2022). factoextra: extract and visualize the results
of multivariate data analyses, the comprehensive R archive network. Available at:
https://cran.r-project.org/package=factoextra (Accessed December 10, 2022).
Khandia, R., Singhal, S., Kumar, U., Ansari, A., Tiwari, R., Dhama, K., et al.
(2019). Analysis of Nipah virus codon usage and adaptation to hosts. Front.
Microbiol. 10, 886–918. doi:10.3389/fmicb.2019.00886
Published by Frontiers
Institute of Virology
23 Biomedical Research Center, Slovak Academy of Sciences
Kanyema et al.
Kumar, N., Kaushik, R., Tennakoon, C., Uversky, V. N., Mishra, A., Sood, R., et al.
(2021). Evolutionary signatures governing the codon usage bias in coronaviruses
and their implications for viruses infecting various bat species. Viruses 13,
1847–1918. doi:10.3390/v13091847
Kumar, N., Kulkarni, D. D., Lee, B., Kaushik, R., Bhatia, S., Sood, R., et al. (2018).
Evolution of codon usage bias in henipaviruses is governed by natural selection and
is host-specific. Viruses 10, 604. doi:10.3390/v10110604
Lobry, J., and Gautier, C. (1994). Hydrophobicity, expressivity and
aromaticity are the major trends of amino-acid usage in 999 Escherichia
coli chromosome-encoded genes. Nucleic Acids Res. 22 (15), 3174–3180.
doi:10.1093/nar/22.15.3174
Loh, J., Zhao, G., Presti, R. M., Holtz, L. R., Finkbeiner, S. R., Droit, L., et al.
(2009). Detection of novel sequences related to african swine fever virus in human
serum and sewage. J. Virology 83 (24), 13019–13025. doi:10.1128/JVI.00638-09
Lubisi, B. A., Bastos, A. D. S., Dwarka, R. M., and Vosloo, W. (2005). Molecular
epidemiology of african swine fever in East Africa. Archives Virology 150,
2439–2452. doi:10.1007/s00705-005-0602-1
Ma, X., Chang, Q., Ma, P., Li, L., Zhou, X., Zhang, D., et al. (2017). Analyses
of nucleotide, codon and amino acids usages between peste des petits
ruminants virus and rinderpest virus. Gene 637, 115–123. doi:10.1016/j.
gene.2017.09.045
Mordstein, C., Cano, L., Morales, A. C., Young, B., Ho, A. T., Rice, A. M.,
et al. (2021). Transcription, mRNA export, and immune evasion shape the
codon usage of viruses. Genome Biol. Evol. 13 (9), evab106–14. doi:10.1093/
gbe/evab106
Mueller, S., Papamichail, D., Coleman, J. R., Skiena, S., and Wimmer, E. (2006).
Reduction of the rate of poliovirus protein synthesis through large-scale codon
deoptimization causes attenuation of viral virulence by lowering specific infectivity.
J. Virology 80 (19), 9687–9696. doi:10.1128/JVI.00738-06
Nakamura, Y., Gojobori, T., and Ikemura, T. (2000). Codon usage tabulated from
international DNA sequence databases: status for the year 2000. Nucleic Acids Res.
28 (1), 292. doi:10.1093/nar/28.1.292
Nasrullah, I., Butt, A. M., Tahir, S., Idrees, M., and Tong, Y. (2015). Genomic
analysis of codon usage shows influence of mutation pressure, natural selection, and
host features on marburg virus evolution. BMC Evol. Biol. 15, 174–215. doi:10.1186/
s12862-015-0456-4
Peng, Q., Ni, Y., Zhang, X., Liu, M., Li, J., Li, B., et al. (2022). Comprehensive
analysis of codon usage patterns of porcine deltacoronavirus and its host
adaptability. Transbound. Emerg. Dis. 2022, e2443–e2455. doi:10.1111/tbed.14588
Pu, F., Wang, R., Yang, X., Hu, X., Wang, J., Zhang, L., et al. (2023). Nucleotide
and codon usage biases involved in the evolution of african swine fever virus: a
comparative genomics analysis. J. Basic Microbiol. 2023, 499–518. doi:10.1002/
jobm.202200624
Puigbò, P., Aragonès, L., and Garcia-vallvé, S. (2010). RCDI/eRCDI: a web-server
to estimate codon usage deoptimization. BMC Res. Notes 3, 87–95. doi:10.1186/
1756-0500-3-87
Puigbò, P., Bravo, I. G., and Garcia-vallve, S. (2008). CAIcal: a combined set of tools
to assess codon usage adaptation. Biol. Direct 8, 38–8. doi:10.1186/1745-6150-3-38
Salguero, F. J. (2020). Comparative pathology and pathogenesis of african swine fever
infection in swine. Front. Veterinary Sci. 7, 282–314. doi:10.3389/fvets.2020.00282
Sang, H., Miller, G., Lokhandwala, S., Sangewar, N., Waghela, S. D., Bishop, R. P.,
et al. (2020). Progress toward development of effective and safe african swine fever
virus vaccines. Front. Veterinary Sci. 7, 84–89. doi:10.3389/fvets.2020.00084
Shackelton, L. A., Parrish, C. R., and Holmes, E. C. (2006). Evolutionary basis of
codon usage and nucleotide composition bias in vertebrate DNA viruses. J. Mol.
Evol. 62, 551–563. doi:10.1007/s00239-005-0221-1
Sharp, P. M., and Li, W.-H. (1986). An evolutionary perspective on synonymous
codon usage in unicellular organisms. J. Mol. Evol. 24, 28–38. doi:10.1007/bf02099948
Sharp, P. M., and Li, W. (1987). The codon adaptation index - a measure of
directional synonymous codon usage bias, and its potential applications. Nucleic
Acids Res. 15 (3), 1281–1295. doi:10.1093/nar/15.3.1281
Shi, S.-L., Jiang, Y.-R., Liu, Y.-Q., Xia, R.-X., and Qin, L. (2013). Selective pressure
dominates the synonymous codon usage in parvoviridae. Virus Genes 46, 10–19.
doi:10.1007/s11262-012-0818-6
Sueoka, N. (1988). Directional mutation pressure and neutral molecular
evolution. Proc. Natl. Acad. Sci. 85, 2653–2657. doi:10.1073/pnas.85.8.2653
Sueoka, N. (1999a). Translation-coupled violation of parity Rule 2 in human
genes is not the cause of heterogeneity of the DNA G + C content of third codon
position. Gene 238, 53–58. doi:10.1016/s0378-1119(99)00320-0
Acta Virologica
10.3389/av.2023.11562
Sueoka, N. (1999b). Two aspects of DNA base composition: G + C content and
translation-coupled deviation from intra-strand rule of A = T and G = C. J. Mol.
Evol. 49, 49–62. doi:10.1007/pl00006534
Sun, J., Zhao, W., Wang, R., Zhang, W., Li, G., Lu, M., et al. (2020). Analysis of the
codon usage pattern of ha and na genes of H7N9 influenza A virus. Int. J. Mol. Sci.
21, 7129–7221. doi:10.3390/ijms21197129
Tao, P., Dai, L., Luo, M., Tang, F., Tien, P., and Pan, Z. (2009). Analysis of
synonymous codon usage in classical swine fever virus. Virus Genes 38, 104–112.
doi:10.1007/s11262-008-0296-z
Teklue, T., Wang, T., Luo, Y., Hu, R., Sun, Y., and Qiu, H. (2020). Generation
and evaluation of an african swine fever virus mutant with deletion of the CD2v
and UK genes. Vaccines 8, 763–817. doi:10.3390/vaccines8040763
Tian, L., Shen, X., Murphy, R. W., and Shen, Y. (2018). The adaptation of codon
usage of + ssRNA viruses to their hosts. Infect. Genet. Evol. 63, 175–179. doi:10.1016/j.
meegid.2018.05.034
Todorov, H., Fournier, D., and Gerber, S. (2018). Principal components analysis:
theory and application to gene expression data analysis. Genomics Comput. Biol. 4
(2), 100041–100111. doi:10.18547/gcb.2018.vol4.iss2.e100041
Tyagi, A., Kumar, B. T. N., and Singh, N. K. (2017). Genome dynamics and
evolution of codon usage patterns in shrimp viruses. Archives Virology 162 (10),
3137–3142. doi:10.1007/s00705-017-3445-7
Urbano, A. C., and Ferreira, F. (2020). Role of the dna-binding protein pa104r in
asfv genome packaging and as a novel target for vaccine and drug development.
Vaccines 8 (4), 585–617. doi:10.3390/vaccines8040585
Wang, G., Xie, M., Wu, W., and Chen, Z. (2021). Structures and functional
diversities of asfv proteins. Viruses 13 (11), 2124. doi:10.3390/v13112124
Wang, H., Liu, S., Zhang, B., and Wei, W. (2016). Analysis of synonymous codon
usage bias of zika virus and its adaption to the hosts. PLoS ONE 11 (11),
01662600–e166322. doi:10.1371/journal.pone.0166260
Wickham, H., Chang, W., Henry, L., Pedersen, T. L, Takahashi, K., Wilke, C.,
et al. (2022). ggplot2: create elegant data visualisations using the grammar of
graphics, the comprehensive R archive network. Available at: https://cran.r-project.
org/package=ggplot2 (Accessed November 10, 2022).
Wold, S., Esbensen, K., and Geladi, P. (1987). Principal component analysis.
Chemom. Intelligent Laboratory Syst. 2, 37–52. doi:10.1016/0169-7439(87)80084-9
Wright, F. (1990). The “effective number of codons” used in a gene. Gene 87,
23–29. doi:10.1016/0378-1119(90)90491-9
Yao, H., Chen, M., and Tang, Z. (2019). Analysis of synonymous codon usage bias
in flaviviridae virus. BioMed Res. Int. 2019, 1–12. doi:10.1155/2019/5857285
Yao, X., Fan, Q., Yao, B., Lu, P., Rahman, S. U., Chen, D., et al. (2020). Codon
usage bias analysis of bluetongue virus causing livestock infection. Front. Microbiol.
11, 655–712. doi:10.3389/fmicb.2020.00655
Yu, X., Liu, J., Li, H., Liu, B., Zhao, B., and Ning, Z. (2021a). Comprehensive analysis of
synonymous codon usage bias for complete genomes and E2 gene of atypical porcine
pestivirus. Biochem. Genet. 59, 799–812. doi:10.1007/s10528-021-10037-y
Yu, X., Liu, J., Li, H., Liu, B., Zhao, B., and Ning, Z. (2021b). Comprehensive
analysis of synonymous codon usage patterns and influencing factors of porcine
epidemic diarrhea virus. Archives Virology 166, 157–165. doi:10.1007/s00705-020-
04857-3
Zhang, J., Wang, M., Liu, W., Zhou, J., Chen, H., Ma, L., et al. (2011). Analysis of
codon usage and nucleotide composition bias in polioviruses. Virology J. 8, 146–148.
doi:10.1186/1743-422X-8-146
Zhang, K., Yang, B., Shen, C., Zhang, T., Hao, Y., Zhang, D., et al. (2022).
MGF360-9L is a major virulence factor associated with the african swine fever virus
by antagonizing the JAK/STAT signaling pathway. mBio 13 (1), 1–19. doi:10.1128/
MBIO.02330-21
Zhang, X., Cai, Y., Zhai, X., Liu, J., Zhao, W., Ji, S., et al. (2018). Comprehensive
analysis of codon usage on rabies virus and other lyssaviruses. Int. J. Mol. Sci. 19,
2397–2415. doi:10.3390/ijms19082397
Zheng, X., Nie, S., and Feng, W. (2022). Regulation of antiviral immune response
by African swine fever virus (ASFV). Virol. Sin. 37 (2), 157–167. doi:10.1016/j.virs.
2022.03.006
Zhou, J., Gao, Z., Sun, D., Ding, Y., Zhang, J., Stipkovits, L., et al. (2013). A
comparative analysis on the synonymous codon usage pattern in viral functional
genes and their translational initiation region of ASFV. Virus Genes 46, 271–279.
doi:10.1007/s11262-012-0847-1
Zhou, J., Zhang, J., Sun, D., Ma, Q., Chen, H., Ma, L., et al. (2013). The distribution
of synonymous codon choice in the translation initiation region of dengue virus.
PLoS ONE 8 (10), 772399–e77247. doi:10.1371/journal.pone.0077239
Published by Frontiers
Institute of Virology
24 Biomedical Research Center, Slovak Academy of Sciences